Method of transffering selected molecules

ABSTRACT

A method of highly efficiently transferring various selected molecules into various cells and a method of fusing cells. Cells and/or selected molecules such as polynucleotide are treated with cold gas plasma to thereby transfer the selected molecules located around cells into the cells, or cells are fused by treating the cells with cold gas plasma. Moreover, an apparatus for transferring selected molecules or fusing cells having a cold gas plasma generation unit for transferring selected molecules into cells is provided.

TECHNICAL FIELD

[0001] The present invention relates to a method of transferring aselected molecule, for example, polynucleotides such as genes, proteins,physiologically-active molecules and others into cells, and a method offusing cells, or to an apparatus for these.

BACKGROUND ART

[0002] In genetic engineering and development of medicines in the fieldof recent medical science, pharmaceutics and others, there is increasingthe necessity of transferring a selected molecule, for example,polynucleotides such as genes, proteins, physiologically-activemolecules, candidates for medicines and others into cells, followed byinspecting the function of the gene in the cells or the physiologicalactivity of the physiologically-active molecule in them. At present, anelectroporation method, a gene gun method, a liposome method, a cellfusion method, a virus vector method and others are employed fortransferring selected molecules, but, in any of these, selectedmolecules could not always be satisfactorily transferred into cells.

[0003] The electroporation method and the gene gun method may apply tomany cells, but they require complicated operations and are difficult tomodify for HTS. The liposome method is problematic in that the cells towhich it may apply are limited. In addition, these methods are allexpensive, and even if they could be modified for HTS, they shall beextremely expensive. Further, most of them are not satisfactory in pointof the transfer efficiency.

[0004] The human and mouse gene arrangements have now been clarified,and it is urgently necessary to analyze the genes having unknownfunctions so as to clarify their functions. In that situation, it isindispensable to develop a gene transfer method enough for HTS. Even inHTS, it is still desired that the method is efficient and enablesfunctional analysis of various genes. For this, it is also urgentlynecessary to develop a high-efficiency gene transfer method not forspecific limited cells but for various cells.

[0005] An object of the present invention is to solve the problems withthe related art as above and to provide a high-efficiency method oftransferring selected molecules into various types of cells and a methodof fusing cells.

DISCLOSURE OF THE INVENTION

[0006] We, the present inventors have found that, when a cell and/or aselected molecule is/are processed with cold gas plasma, then theselected molecule existing around the cell is transferred into the cell,and have completed the present invention. Further, we have found that,when cell is processed with cold gas plasma, then they are fused.

[0007] Specifically, the invention is as follows:

[0008] [1] A method of transferring a selected molecule into a cell,which comprises processing a cell and/or a selected molecule with coldgas plasma to thereby transfer the selected molecule existing around thecell into the cell.

[0009] [2] The selected molecule transfer method of [1], wherein theselected molecule is previously made to exist around the cell, and thecell is then processed with cold plasma.

[0010] [3] The selected molecule transfer method of [1] or [2] whereinthe selected molecule is polynucleotide.

[0011] [4] A method of fusing cells, which comprises processing cellswith plasma.

[0012] [5] An apparatus for processing a target, which is a cell and/orselected molecule, with cold gas plasma to thereby transfer the selectedmolecule existing around the cell into the cell, or for processing cellswith plasma to thereby fuse the cells.

[0013] [6] The apparatus in [5] for transferring a selected molecule isequipped with cold gas plasma generation unit comprising open-airdischarge generate the cold gas plasma

[0014] The selected molecules as referred to herein are molecules thatare selected so as to be transferred into the intended cells. Theselected molecules include high-molecular compounds, low-molecularphysiologically-active substances, and candidates for medicines, forexample, polynucleotides such as DNA, RNA and their derivatives, andproteins such as signal transfer proteins, transcriptional controlfactors and their derivatives. Of those selected molecules, preferredare polynucleotides and their derivatives.

[0015] The cells as referred to herein are the intended cells into whichthe selected molecules are transferred, and they are not specificallydefined. Examples of the cells are procaryotic cells such as Escherichiacoli, actinomycetes, Bacillus subtilis; and eucaryotic cells such asyeast, animal cells and vegetable cells. In addition, those having alipid bilayer structure such as erythrocyte ghosts and liposomes arealso within the scope of the cells in the invention.

[0016] For increasing the transfer efficiency of selected molecules intothese cells, it is possible to use cells that are formed in accordancewith an ordinary method of forming competent cells for gene transferthereinto. If desired, the method of the invention may be combined withany other gene transfer method such as a liposome method of usingcationic lipid, e.g., lipofectamin (GIBCO-BRL) or liposome, whereby theefficiency of the method of transferring selected molecules into thecells may be further increased.

[0017] The cold gas plasma (cold non-equilibrium plasma, coldweakly-ionized plasma) for use in the invention may be generated, forexample, through corona discharging, and its properties may be varieddepending on the type and the condition of the generation unit for it.Regarding the plasma that is used for carrying out the invention, thetype of the gas for it, the plasma density, the electron temperature andthe processing time with it may be suitably determined depending on thecells to be used and the selected molecules and also on the environmentin which the operation is effected. Regarding the type of the gas to beused for the cold gas plasma, at least one selected from a groupconsisting of oxygen, air, carbon dioxide, nitrogen and argon ispreferred.

[0018] The cold gas plasma generation unit to be used in the inventionmay be an open or closed unit. In view of the easiness with it inprocessing cells, preferred is an open unit.

[0019] A specific example of the apparatus of the invention is shown inFIG. 1. This apparatus comprises an electric line and a gas line. Theelectric line comprises a signal generator, a linear amplifier, amatching circuit and a booster transformer, and these act to control theparameters of inter-electrode voltage, inter-electrode distance,frequency, pulse period, duty and others. Under the controlledcondition, the head is discharged to generate various plasmas. On theother hand, in the gas line, a single or mixed gas fed from a gascylinder or an air pump passes through a needle valve to have asuitably-controlled flow rate, and fed to the head. The plasma havingbeen generated in the head is blown out by the gas toward the samplethat is set in front of the head. In order that the plasma irradiationmay be effected in suitable conditions for every sample of cells,different types of organisms or selected molecules (including genes,low-molecular substances, proteins and others) , these conditions may bevaried to change the determination of the conditions for the plasma andits irradiation.

[0020] One specific embodiment of the invention comprises removing theculture from the cultured adhesive cells that have been cultured on acell culture kit such as a plate, or from the cells that have beencollected through centrifugation, filtration or the like, followed byadding a small amount of a solution of a selected molecule to thesurface of each cell. Next, this is processed with plasma that has beengenerated by a plasma generator. The plasma processing time may fallgenerally between a few seconds and tens seconds, through varyingdepending on the plasma condition. After the plasma treatment, a mediumis added to the cells and the cells are further cultured therein. Incase where the selected molecule is a vector that contains a gene or thelike, this is effective for gene recombination experiments. In casewhere the selected molecule is a candidate substance for a medicine thatis targeted to a specific molecule in cells, this is effective forscreening the candidates.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 is a schematic view showing an apparatus for gene transferor cell fusion in the invention.

[0022]FIG. 2 is a schematic view showing the relationship between theplasma generator unit (electrode part) in the apparatus for genetransfer or cell fusion in the invention, and the sample to be processedwith the unit.

[0023]FIG. 3, FIG. 4 and FIG. 5 each show the spectral data of theplasma having been generated under different conditions in the apparatusused for gene transfer or cell fusion in the Examples of the invention.For measuring the data, a spectrophotometer, Otsuka Electronics'MCPD-3000 was used. Separated by about 3 cm from the plasma, a probe wasset to receive the spectrum of the plasma.

BEST MODES OF CARRYING OUT THE INVENTION

[0024] The invention is described more concretely with reference to thefollowing Examples, to which, however, the invention should not belimited.

EXAMPLE 1

[0025] As in FIG. 1, an apparatus of the invention was constructed. Thecondition of the plasma generator in this apparatus is as follows: Theinter-electrode voltage is from a few kV to more than ten kV; theinter-electrode distance is from 10 to 15 mm; the frequency is from 20to 40 kHz; the pulse period is from 30 to 90 Hz; the duty is from 25 to100%; the gas is any of air, nitrogen, oxygen, carbon dioxide, argon, orhelium/air=1/1, and its irradiation time is approximately from 1 to 5seconds. The distance between the electrode and the sample isapproximately from 1 to 3 cm. Within the range, a suitable condition forthe sample was selected.

[0026] In this apparatus, for example, plasma having the spectrum as inFIG. 3 to FIG. 5 can be generated. Using the plasma generationapparatus, gene transfer was carried out herein.

[0027] Chinese hamster pulmonary fibroblasts, CHL cells were sowed in acell culture plate having a diameter of 60 mm, and cultured overnight at37° C. under the condition of 5% carbon dioxide. The number of the cellsfirst sowed at the start of the culture was 1×10⁶ cells/well. After itwas confirmed that the cells well adhered to the plate, the medium wasremoved from the culture plate, and 110 μl of a GFP expression plasmidliquid (1 μg/μl) was added to the cell surface. Then, this was subjectedto plasma irradiation under various conditions with the apparatus of theinvention. Immediately after the plasma irradiation, a medium was addedthereto and the cells were cultured overnight. Then, the cells wereobserved with a fluorescence microscope for expression of GFP protein,and the number of the expressing cells per the field of view wascounted. In addition, the GFP protein-expressing cells per all the cellsin every well were quantified through FACS flow cytometry.

[0028] As a result, GFP expression was observed under every condition.The transfer efficiency was at most about 60% in microscopic observationand was approximately from 5 to 25% in FACS inspection.

[0029] A plasma generator, Plasma Surface Treater ST-7000, sold on themarket by KEYENCE is an apparatus of the same type as that of theapparatus used herein. This may generate plasma having the spectrum asin FIG. 5, and this may be usable for the object of the invention.

EXAMPLE 2

[0030] 2-1) Transfer into adhesive cells of established animal cellline:

[0031] Established animal cells were sowed in a 6-well plate andcultured overnight at 37° C. under the condition of 5% carbon dioxide.The number of the cells, Chinese hamster fibroblasts CHL cells firstsowed at the start of the culture was 1×10⁶ cells/well; and that ofhuman uterine cancer-derived Hela cells was 2.5×10⁵ cells/well. After itwas confirmed that the cells well adhered to the plate, the medium wasremoved from the culture plate, and 50 μl of a GFP expression plasmid(pEGFP-C1) liquid (1 μg/μl) was added to the cell surface. Then, thiswas subjected to plasma irradiation with a plasma generator. Thecondition of the plasma generator was as follows: The inter-electrodevoltage during discharge was from 10 to 14 kV; the inter-electrodedistance was 13 mm; the frequency was 23.3 kHz; the pulse period was 60Hz; the duty was 50%; and the gas, air flow rate was 38 liters/min. Theirradiation was effected for about 3 seconds. The distance between theelectrode and the sample was suitably determined. Immediately after theplasma irradiation, a medium was added thereto and the cells werecultured overnight. Then, the cells were observed with a fluorescencemicroscope for expression of GFP protein, and the number of theexpressing cells per the field of view was counted. In addition, the GFPprotein-expressing cells per all the cells in every well were quantifiedthrough FACS flow cytometry.

[0032] As a result, GFP expression was observed in all types of thecells. Under the condition that the inter-electrode voltage duringdischarge was from 10 to 14 kV, the inter-electrode distance was 13 mm,the frequency was 20 kHz; the pulse period was 60 Hz; the duty was 50%;and the gas, air flow rate was 38 liters/min, the transfer efficiencywas at most about 70% in microscopic observation and was about 30% inFACS inspection (this is the gene transfer efficiency per the number ofall the cells in the dish). It is said that the transfer efficiency withHela cells is low in existing methods, but the method of the inventionhas made it possible to attain a high transfer efficiency of about 70%or so in some region.

EXAMPLE 3

[0033] Transfer into planktonic cells of established animal cell line:

[0034] A phosphate buffer suspension of human acute lymphoblasticleukemic peripheral blood-derived T cell-line Jurkat cells, 2.25×10⁷cells/ml, was prepared. 25 μl of the cell suspension was mixed with thesame amount of a GFP expression plasmid solution (2 μg/μl), and theresulting mixture was put into a fresh 6-well plate. Its amount was 50μl/well. This was spread thin in the bottom of each well, and thenirradiated with plasma with a plasma generator. In this Example, theplasma generator was driven as in Example 1. Immediately after theplasma irradiation, a medium was added thereto and the cells werecultured overnight at 37° C. under the condition of 5% carbon dioxide.Then, the cells were observed with a fluorescence microscope forexpression of GFP protein. In addition, the GFP protein-expressing cellsper all the cells in every well were quantified through FACS flowcytometry.

[0035] As a result, GFP protein expression was confirmed in observationwith a fluorescence microscope. The transfer efficiency confirmedthrough FACS was about 25%.

EXAMPLE 4

[0036] Transfer into rat cerebral cortex cells:

[0037] A rat cerebral cortex was prepared as follows: A pregnant rat(Wister, 17-day pregnant) was anesthetized and its uterus with its fetustherein was taken out through abdominal operation into L-15 (GIBCO-BRL).Then, the cerebral cortex site was separated from the whole brain of thefetus. 40 ml of 0.25% trypsin solution and 80 μl of 1% DNAse solutionwere added to the cerebral cortex site, which was then incubated at 37°C. for 20 minutes. The resulting supernatant was removed, and 10 ml ofFBS was added to the residue. This was pipetted to loosen the cells.This was passed through a cell strainer, and about 20 ml of a Neurobasalmedium (GIBCO-BRL) was added thereto, and the cells were collectedthrough centrifugation. The cells were suspended in a Neurobasal mediumfor culture initiation (25 μM glutamic acid, 500 μM glutamine, 30 nMNaSeO₃, containing penicillin and streptomycin) to be 5×10⁵ cells/ml,and these were cultured in a 6-well plate at 37° C. under the conditionof 5% carbon dioxide.

[0038] After it was confirmed that the cells well adhered to the plate,the medium was removed from the culture plate, and 50 μl of a GFPexpression plasmid liquid (1 μg/μl) was added to the cell surface. Then,this was subjected to plasma irradiation with a plasma generator. Thecondition of the plasma generator was as follows: The inter-electrodevoltage during discharge was from 10 to 14 kV; the inter-electrodedistance was 13 mm; the frequency was 23.3 kHz; the pulse period was 60Hz; the duty was 50%; and the gas, air flow rate was 38 liters/min. Theirradiation was effected for about 1 second. Immediately after theplasma irradiation, a medium was added thereto and the cells werecultured overnight. Then, the cells were observed with a fluorescencemicroscope for expression of GFP protein.

[0039] As a result, GFP protein-expressing cells were found. Thetransfer efficiency was at most about 70% per field of view. Genetransfer into the primary-cultured animal cells as herein was almostimpossible in existing gene-transfer methods. The method of the presentinvention has enabled safe, simple and efficient gene transfer into theprimary-cultured animal cells.

EXAMPLE 5

[0040] Transfer into rat cerebellar granular cells:

[0041] A 9-day-old Wister rat was anesthetized with ether and its brainwas taken out. The cerebellum was cut out of it, and its meninx wasremoved. The cerebellum was cut into pieces, and 10 ml of a papainsolution (this was prepared by mixing 90 units of papain(Worthington-biochem) in 10 ml of a phosphate buffer with 2 mg ofDL-cysteine (Sigma) and 50 mg of albumin dissolved therein, leaving theresulting mixture at 37° C. for a while to activate it, then addingthereto 50 μl of DNase I (Takara) , and before use, this was filteredand sterilized) was added thereto, and shaken at 37° C. for about 30minutes. 6 ml of horse serum (HS) was added to it and centrifuged. Theresulting tissue deposit was suspended in a serum medium (5% PFCS and 5%HS containing DME/F12 (1/1) medium). This was sowed in apolyethylenimine-coated 6-well plate to be 2.5×10⁶ cells/well, andcultured overnight at 37° C. under the condition of 5% carbon dioxide.With that, the medium was exchanged with a high-potassium (26 mM), 1 μMAraC (Sigma)—containing medium (5% HS, potassium bicarbonate/2.1 g, 30nMNa₂SeO₄in MEM (Sigma)), and the cells were further cultured for 5 daysat 37° C. under the condition of 5% carbon dioxide.

[0042] After it was confirmed that the cells well adhered to the plate,the medium was removed from the culture plate, and 50 μl of a GFPexpression plasmid liquid (1 μg/μl) was added to the cell surface. Then,this was subjected to plasma irradiation with a plasma generator. Thecondition of the plasma generator was as follows: The inter-electrodevoltage during discharge was from 10 to 14 kV; the inter-electrodedistance was 13 mm; the frequency was 23.3 kHz; the pulse period was 60Hz; the duty was 50%; and the gas, air flow rate was 38 liters/min. Theirradiation was effected for about 1 second. Immediately after theplasma irradiation, a medium was added thereto and the cells werecultured overnight. Then, the cells were observed with a fluorescencemicroscope for expression of GFP protein.

[0043] As a result, GFP protein-expressing cells were found. Thetransfer efficiency was at most about 40% per field of view. Genetransfer into the primary-cultured animal cells as herein was almostimpossible in existing gene-transfer methods. The method of the presentinvention has enabled safe, simple and efficient gene transfer into theprimary-cultured animal cells.

EXAMPLE 6

[0044] Transfer into human umbilical vein-derived hemal endothelialcells (HUVEC):

[0045] Cells were cultured, using Total Kit (Toyobo) for normal humanumbilical vein endothelial cells.

[0046] Normal human umbilical vein endothelial cells (HUVEC) were sowedin a 6-well plate to be 2.5×10⁵ cells/well, and cultured overnight at37° C. under the condition of 5% carbon dioxide. After it was confirmedthat the cells well adhered to the plate, the medium was removed fromthe culture plate, and 50 μl of a GFP expression plasmid liquid (1μg/μl) was added to the cell surface. Then, this was subjected to plasmairradiation with a plasma generator. The condition of the plasmagenerator was as follows: The inter-electrode voltage during dischargewas from 10 to 14 kV; the inter-electrode distance was 13 mm; thefrequency was 23.3 kHz; the pulse period was 60 Hz; the duty was 50%;and the gas, air flow rate was 38 liters/min. The irradiation waseffected for about 1 second or 3 seconds. Immediately after the plasmairradiation, a medium was added thereto and the cells were culturedovernight. Then, the cells were observed with a fluorescence microscopefor expression of GFP protein.

[0047] As a result, GFP gene transfer was confirmed also into the humanumbilical vein-derived hemal endothelial cells. The transfer efficiencywas at most about 50% per field of view. Gene transfer into theprimary-cultured animal cells as herein was almost impossible inexisting gene-transfer methods. The method of the present invention hasenabled safe, simple and efficient gene transfer into theprimary-cultured animal cells.

EXAMPLE 7

[0048] Differentiation induction of PC12 cells into sympathetic nervoussystem:

[0049] This is to investigate as to whether or not the cells processedaccording to the method of the invention for gene transfer thereintocould still maintain their function. Concretely, PC12 cells were testedfor differentiation potency into sympathetic nervous systems.

[0050] A GFP gene was transferred into PC12 cells according to themethod of the invention as in Example 1. NGF was added to the culture ofthe cells to be 100 ng/ml, and the cells were cultured for 6 days. Then,the morphology of the PC12 cells that had been confirmed to haveexpressed GFP protein with a fluorescence microscope was observed tocheck them for differentiation potency into sympathetic cells.

[0051] As a result, the PC12 cells having the GFP gene transferredthereinto according to the method of the invention still expressed GFPprotein even in 6 days after the NGF addition thereto, and in addition,the cell morphology observation surely supported neural processextension from the cells. This confirms that the gene transfer methodassisted by plasma irradiation does not change the property intrinsic toPC12 cells for differentiation potency with NGF into sympathetic cells.

EXAMPLE 8

[0052] CREB activation through gene transfer into PC12 cells (reportergene assay):

[0053] This is to investigate as to whether or not the gene having beentransferred into cells according to the method of the invention couldexhibit its function in the cells. Concretely, PC12 cells with CREB andPKA transferred thereinto were analyzed through reporter gene assay asto whether or not CREB therein could be activated by the PKA gene alsotherein.

[0054] Rat pheochromocytoma PC12 cells (ATCC No. CRL-1721) were sowed ina collagen IV-coated 6-well plate to be 1×10⁶ cells/well, and culturedovernight at 37° C. under the condition of 5% carbon dioxide. After itwas confirmed that the cells well adhered to the plate, the medium wasremoved from the culture plate. Then, the cells were processed with amixture that had been prepared by mixing a PKA gene (pFC-PKA) (1 μg/μl),a CREB reporter gene (pCRE-Luc) with a luciferase gene linked downstreamthe response sequence of activated CREB (1 μg/μl) and an internalstandard, Renilla luciferase gene (pRL-SV40) of 17 μl each, according tothe method of the invention as in Example 2 to thereby co-transfer thegenes into the PC12 cells. One day after the gene transfer, the PC12cells were checked for luciferase activity.

[0055] As a result, significant luciferase activity increase, or thatis, significant CREB transcription activity increase was found in thePKA-transferred cells as compared with the control vector-transferredcells. This confirms that the gene having been transferred into PC12cells of an established culture cell line according to the gene transfermethod associated with plasma irradiation exhibits its function in thecells.

EXAMPLE 9

[0056] CREB activation through gene transfer into rat cerebellargranular cells (reporter gene assay):

[0057] This is to investigate as to whether or not the same result asabove could be obtained even in primary-cultured animal cells into whichgene transfer is difficult according to conventional methods. For this,rat cerebellar granular cells were tried in the same manner as inExample 8.

[0058] Rat cerebellar granular cells were established in the same manneras in Example 5. A PKA gene (pFC-PKA) (1 μg/μl), a CREB reporter gene(pCRE-Luc) with a luciferase gene linked downstream the responsesequence of activated CREB (1 μg/μl) and an internal standard, Renillaluciferase gene (pRL-SV40) were co-transferred into the cells by the useof a plasma generator as in Example 2, and the cells were then culturedfor 1 day. Then, the cells were checked for luciferase activity.

[0059] As a result, the luciferase activity of the primary ratneurocytes significantly increased as compared with the controlgene-transferred cells. Accordingly, this confirms that, even in primaryrat neurocytes (rat cerebellar granular cells) , the plasma transfermethod gives no abnormality to the CREB signal transfer pathway by PKA.

[0060] From the above, it has been clarified that the gene transfermethod associated with plasma irradiation makes it possible to transfera gene even into primary neurocytes, into which gene transfer isdifficult in conventional methods, and the transferred gene can expressits function in the cells, and that the method simplifies reporter geneassay.

EXAMPLE 10

[0061] Apoptosis induction through BAD gene transfer into rat cerebellargranular cells:

[0062] Rat cerebellar granular cells were prepared in the same manner asin Example 5. 5 days after AraC addition thereto, the medium was removedfrom the cells. A solution of an apoptosis-inducing gene, BAD gene(pcDNA3.1/GS-BAD) (1 μg/50 μl) was added to the cells to be 50 μl/well,and the cells were subjected to plasma irradiation under the samecondition as in Example 2. Immediately after this, a medium was addedthereto, and the cells were cultured overnight at 37° C. under thecondition of 5% carbon dioxide. With that, the caspase activity of thecells was measured with CaspASE FITC-VAD-FMK in-situ Marker (Promega) toinvestigate as to whether or not apoptosis was induced in the cells.

[0063] As a result, a significant caspase activity was found in the BADgene-transferred cells, as compared with the MOCK cells into which thevector alone was transferred. Namely, apoptosis-induced cells weredetected.

[0064] In addition, the cells were further checked for DNA fragmentationby the use of APO-DIRECT (Pharmingen) for the purpose of confirming asto whether the cells, into which the BAD gene had been transferredaccording to the method of the invention, were surely induced toapoptosis.

[0065] As a result, cells with significant DNA fragmentation weredetected in the BAD gene-transferred cells as compared with the MOCKcells with the vector alone transferred thereinto.

[0066] The result confirms that the BAD gene having been transferredinto rat cerebellar granular cells according to the method of theinvention significantly induced apoptosis in the cells and exhibited itsfunction. From the above, it has been confirmed that the genetransferred into primary culture cells according to the method of theinvention surely exhibits its function in the cells.

EXAMPLE 11

[0067] Investigation of cell fusion with Chinese hamster lung-derivedCHL cells, human uterus cancer-derived Hela cells, and ratpheochromocytoma PC12 cells:

[0068] In the same manner as in Example 2, a GFP gene was transferredinto CHL cells and into Hela cells. These cells were checked for fusedcells, using a fluorescence microscope. The cells were stained with DifQuick (International Reagents) on their nuclei, and the situation of thefused cells was observed with a microscope. The nuclei were stained asfollows: After processed with plasma, the cells were cultured for oneday, and the culture liquid was absorbed away from the cells. Then, thecells having adhered to the dish were washed with a phosphate buffer,and about 5 ml/well of methanol (Wako) was added thereto, and the cellswere fixed for 5 minutes at room temperature. About 2 ml/well ofstaining liquid I was added to them, and this was immediately absorbedaway. Next, about 2 ml/well of staining liquid II was added thereto, andthis was also immediately absorbed away. The adhesive cells were washeda few times with ion-exchanged water, and then observed with amicroscope.

[0069] In the CHL and Hela cells with a GFP gene transferred thereintoaccording to the method of the invention, fused large cells were found.When the nucleus of each cell processed through plasma irradiation wasstained, it was found that some cells were fused along with their nucleiand some others were fused to give polynuclear cells.

[0070] On the other hand, a GFP gene was transferred into ratpheochromocytoma PC12 cells in the same manner as in Example 1, forwhich, however, the number of the cells sowed in the 6-well plate was5×10⁶/well and was relatively large. NGF was added to the cells in thesame manner as in Example 7, and the cells were checked fordifferentiation potency.

[0071] Even the fused cells expressed GFP protein in 6 days after theNGF addition, and neural process extension was surely found from thefused cells. This suggests that the fused cells formed through plasmairradiation also do not change the property intrinsic to PC12 cells fordifferentiation potency with NGF into sympathetic cells.

EXAMPLE 12

[0072] Combination with liposome method:

[0073] This is to investigate as to whether or not the method of theinvention enables gene transfer into cells even under difficultconditions for liposome-assisted gene transfer.

[0074] In the liposome method, a liposome reagent and a transfer plasmidare mixed to prepare mixed particles having a suitable size, and theparticles are introduced into cells. Accordingly, in the method, apredetermined number of particles having a predetermined size must beprepared. This Example is to investigate as to whether or not the genetransfer efficiency could be improved under more difficult conditionshaving a higher plasmid concentration and a higher liposome reagentcondition than in the optimized conditions. In this, a GFP gene wastransferred into cells.

[0075] Concretely, 100 μg of a GFP expression plasmid and 100 μl ofLipofectin 2000 (GIBCO-BRL) were previously mixed and left at roomtemperature for 15 minutes to prepare a plasmid/Lipofectin conjugate.From the previously day, CHL cells were kept cultured in a 6-well plate,and the culture supernatant was removed from the plate. With that, 200μl of the plasmid/Lipofectin conjugate was added to the plate. Using aplasma generator, this was irradiated with plasma. The condition was asfollows: The frequency was 23.3 kHz, the pulse period was 60 Hz, theduty was 50%, and the gas, air flow rate was 38 liters/min. Immediatelyafter the plasma irradiation, a medium was added thereto, and the cellswere cultured at 37° C. under the condition of 5% carbon dioxide. Aftercultured for one day, the cells were observed with a fluorescencemicroscope, and the GFP protein-expressing cells were detected throughFACS.

[0076] Even in this condition, a few cells were found to have GFPtransferred thereinto in a liposome method alone. In the combinationwith the method of the invention, the gene-transferred cells in theplasma irradiation range increased as compared with the liposome methodalone. In addition, the ratio of the gene-transferred cells to all thecells in each well was obtained through FACS. It was found that thetransfer efficiency in the combination with the method of the inventionincreased by about 1.6 times as compared with the liposome method alone.Accordingly, when the method of the invention is combined with any othergene transfer method, or when a liposome or any other carriersubstitutable with it and capable of promoting plasmid transfer intocells is bound for use in the method of the invention, the transferefficiency could be improved even in difficult cells and under difficultconditions in and under which the transfer efficiency has heretoforebeen not good.

EXAMPLE 13

[0077] In this, a plasma generator was tested for GFP-expression plasmidtransfer into cultured cells.

[0078] The method is as follows: Rat pheochromocytoma PC12 cells (ATCCNo. CRL-1721) were sowed in a collagen IV-coated 6-well plate to be1×10⁶ cells/well, and cultured overnight. After it was confirmed thatthe cells well adhered to the plate, the medium was removed from theculture plate, and 50 μl of a GFP expression plasmid liquid (1 μg/μl)was added to the cell surface. Then, this was subjected to plasmairradiation with a plasma generator, Plasma Suface Treater ST-7000 (byKEYENCE). Regarding the condition for the plasma generator ST-7000, thefrequency was any of three conditions of high, low or metal, and theirradiation continued for about 5 seconds in every case. Immediatelyafter the plasma irradiation, a medium was added thereto and the cellswere cultured overnight. Using a fluorescence microscope, the cells werechecked for expression of GFP protein therein.

[0079] The result is as follows: In every condition, GFP expression wasfound in the cells.

[0080] The transfer efficiency was the highest in the high frequencycondition, falling between 50 and 60%.

EXAMPLE 14

[0081] Evans blue (10 mg/ml physiological saline) was thinly applied toall over the abdomen of a nude mouse (8 weeks-old, female, NipponCharles River) in anesthetization. Using a plasma generator, this wasirradiated with plasma. The inter-electrode voltage during discharge wasfrom 10 to 14 kV; the inter-electrode distance was 13 mm; the frequencywas 23.3 kHz; the pulse period was 60 Hz; the duty was 50%; and the gas,air flow rate was 38 liters/min. The irradiation was effected for 2.5seconds. The distance between the electrode and the abdomen was fromabout 24 mm in the remotest to about 18 mm in the nearest. This was leftas such for about 3 minutes, and then the Evans blue-applied site wasfully wiped with absorbent cotton wetted with water and then absorbentcotton wetted with 70% ethanol. With that, the skin was checked for dyedeposition. Thus processed, the mouse was compared with anothersubjected to plasma irradiation alone and with still another coated withEvans blue but not subjected to plasma irradiation.

[0082] As a result, no dye deposition was found in both the mousesubjected to plasma irradiation alone and the mouse coated with Evansblue but not subjected to plasma irradiation. As opposed to these, themouse coated with Evans blue and subjected to plasma irradiation hadsome blue spots in the plasma-irradiated region of the abdomen thereof.Even after about 24 hours, the spots were still seen. Accordingly, it isbelieved that substances of which the molecular weight is on the levelof Evans blue could be transferred into animal individuals throughplasma irradiation.

[0083] Heretofore, a report has been announced, saying that dye ispercutaneously transferred into animal individuals through irradiationwith cold ultrasonic waves (Katsuro Tachibana; the Medical Department ofFukuoka University/the Society of Molecular Biology of Japan, 2001). Thepresent invention suggests that substance transfer into animalindividuals is also possible through plasma irradiation.

INDUSTRIAL APPLICABILITY

[0084] The present invention has enabled high-efficiency transfer of avariety of selected molecules into a variety of cells. It has enabledcell fusion as well. In addition, the invention does not require anycomplicated operation of applying an electric field to every one sampleby the use of electrodes as in electroporation, and does not require anylarge-scale apparatus such as gene guns.

1. A method of transferring a selected molecule into a cell, whichcomprises processing a cell and/or a selected molecule with cold gasplasma to thereby transfer the selected molecule existing around thecell into the cell.
 2. The selected molecule transfer method as claimedin claim 1, wherein the selected molecule is previously made to existaround the cell, and the cell is then processed with cold gas plasma. 3.The selected molecule transfer method as claimed in claim 1 or 2,wherein the selected molecule is polynucleotide.
 4. A method of fusingcells, which comprises processing cells with plasma.
 5. An apparatus forprocessing a target, which is a cell and/or selected molecule, with coldgas plasma to thereby transfer the selected molecule existing around thecell into the cell, or for processing cells with plasma to thereby fusethe cells.
 6. The apparatus in claim 5 for transferring a selectedmolecule, which is equipped with cold gas plasma generation unitcomprising open-air discharge.